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Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H

Abstract

Numerous studies have shown that ribonucleoside monophosphates (rNMPs) are probably abundant among all nonstandard nucleotides occurring in genomic DNA. Therefore, it is important to understand to what extent rNMPs may alter genome integrity and what factors affect their stability. We developed oligonucleotide-driven gene correction assays in Escherichia coli and Saccharomyces cerevisiae to show that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis and produce a genetic change. We discovered that isolated mispaired rNMPs in chromosomal DNA are removed by the mismatch repair system in competition with RNase H type 2. However, a mismatch within an RNA-DNA heteroduplex region requires RNase H type 1 for removal. In the absence of mismatch repair and RNases H, ribonucleotide-driven gene modification increased by a factor of 47 in yeast and 77,000 in E. coli.

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Figure 1: Diagrams and sequences of the loci targeted by the RNA-containing oligonucleotides.
Figure 2: RNase HII cleavage specificity.

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Acknowledgements

The authors thank P.W. Doetsch and his group, and Y.W. Kow for discussions and comments. We are grateful to G.F. Crouse and R. Pai for suggestions on the paper, L.D. Williams for technical support for the gels, C. Flood for technical assistance and all the members of the Storici laboratory for advice in the course of the study. This research was supported by Georgia Cancer Coalition grant R9028 (F.S.), National Science Foundation grant MCB-1021763 (F.S.) and Integrative Biosystems Institute grant IBSI-4 (F.S.).

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Contributions

Y.S. conducted most of the experiments on E. coli, all yeast experiments and statistical analyses of the data. K.D.K. carried out the RNase HII cleavage experiments, analyzed biochemical data and helped with the E. coli experiments. B.W. helped to design the experiments, conducted initial tests on E. coli and analyzed data. F.S. designed most of experiments, analyzed data and wrote the manuscript, with input from all authors.

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Correspondence to Francesca Storici.

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The authors declare no competing financial interests.

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Shen, Y., Koh, K., Weiss, B. et al. Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H. Nat Struct Mol Biol 19, 98–104 (2012). https://doi.org/10.1038/nsmb.2176

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